Method for diagnosing early and late lyme borreliosis

ABSTRACT

The present invention relates to a method for detecting  Borrelia burgdorferi  sensu lato infection or the presence of antibodies against  Borrelia  species in a body fluid from a suspected infected or vaccinated human or other mammal, to a diagnostic kit useful in said method and to an immunoassay method for diagnosing early and late Lyme borreliosis, especially for diagnosing erythema migrans. The methods according to the invention are characterized in that recombinant BBK32 proteins, optionally other immunogenic borrelial proteins, or their fragments are used as antigens.

FIELD OF THE INVENTION

The present invention relates to a method for detecting Borreliaburgdorferi sensu lato infection or the presence of antibodies againstBorrelia species in a body fluid from a suspected infected or vaccinatedhuman or other mammal, to a diagnostic kit useful in said method and toan immunoassay method for diagnosing early and late Lyme borreliosis(LB), especially for diagnosing erythema migrans (EM). The methodsaccording to the invention are characterized in that recombinant BBK32proteins, other immunogenic borrelial proteins and/or their fragmentsare used as antigens.

BACKGROUND OF THE INVENTION

LB is a tick-transmitted spirochetal infectious disease, caused byBorrelia burgdorferi, which is characterized by multistage skin, joint,neurologic and cardiac manifestations [1]. The diagnosis of LB is basedon clinical evaluation of the patients, but serologic assays, mostfrequently the enzyme-linked immunosorbent assay (ELISA) and Westernblotting (WB), are often used to provide supporting evidence ofinfection with B. burgdorferi. In the current routine LB serodiagnostictests, the antigens predominantly used are borrelial flagellin proteinor whole-cell lysate (WCL) of the in vitro-cultured microbes. A two-stepapproach with ELISA and a confirmatory WCL WB has been recommended bythe Centers for Disease Control (ISA) for positive or borderline results[2]. Especially in Europe the applicability of this procedure hasremained questionable [3], mainly due to the existence of three speciesof B. burgdorferi sensu lato causing human LB, B. burgdorferi sensustricto, B. afzelii, and B. garinii [4]. One of the main reasons forthese problems is the antigenic diversity due to variations in thesequences and expression of immunogenic proteins in Several otherdifficulties complicate current LB serology. In LB patients with earlymanifestations, e.g. erythema migrans (EM) and facial palsy, antibodyresponses to the current antigens may be weak or delayed [6]. EM, whichappears at the site of the tick bite days to weeks after exposure, isthe earliest and most common manifestation of LB. Tick bites may easilybe unrecognised, and the clinician has to rely on the appearance of theskin lesion. In a routine clinical setting, EM is considered to bepathognomonic for early LB. The classical appearance of EM is anenlarging, ring-like erythema with a central clearing. However, early inthe course of LB, atypical lesions may occur and cause diagnosticproblems. Recent reports from the US and Europe have shown that duringthe first few days of infection the pathognomonic expanding peripheralerythema with a central clearing appears to be far less common thanlesions with homogeneous redness [33, 34]. The gold standard for thediagnosis of early LB is microbiologic confirmation of LB by culturefrom biopsies taken from EM lesions and/or from blood. PCR-based methodscan also be used to detect B. burgdorferi DNA in skin biopsies. However,in routine clinical practice, these methods are not feasible. Serologicconfirmation of early LB is also problematic due to sensitivity problemsof the current serodiagnostic assays. The sensitivity of the IgM or IgGenzyme-linked immunosorbent assay (ELISA) using borrelial flagella orwhole-cell lysate antigens seldom exceeds 40-50% [6, 25]. Even at latestage LB, up to 5-10% of patients may not have elevated antibody levels[7]. Further, viral infections cause false positivity in several LBtests for IgM antibodies [8]. Furthermore, in a subgroup of patientsafter a successful treatment of LB, antibody levels may stay high evenfor prolonged periods.

The expression of borrelial proteins also varies at different stages inthe life cycle of Borrelia in ticks and in the mammalian hosts. Severalgenes, e.g. bbk32, bbk50, vls and ospE/F homologs [9-11], have beenshown to be selectively expressed in vivo. Moreover, bbk32 expression isdetectable in spirochetes during tick feeding even before transmissionto the host but not in unfed ticks [12]. In two previous studies,antibodies to BBK32 were observed in the sera of B. burgdorferi sensustricto-infected mice and human patients with disseminated LB [9, 13].So far, antigenic properties of the BBK32 proteins in other species ofB. burgdorferi sensu lato are not known.

The application of specific recombinant proteins of B. burgdorferi, suchas outer surface protein A (OspA), OspB, OspC, OspE, OspF, P22, P39,P100, VlsE, and flagellin have improved the performance of ELISA assays[14-16]. However, the immune responses of patients infected withdifferent B. burgdorferi subspecies vary greatly, and certain antigensmay not always be expressed in hosts or be recognized immunologically.Thus, the sensitivity with single recombinant antigens has remainedinsufficient, so far. Another approach to improve the serodiagnosis ofLB has been the use of peptides from certain borrelial proteins asantigens, e.g. a C-terminal de-capeptide from OspC [17] and a peptidecorresponding to a central invariable region in the VIsE protein,invariable region 6, IR6 [18]. Very recently, chimeric proteinscomprising of epitopes of OspA, OspB, OspC, flagellin, and p93 have alsobeen suggested for serodiagnostic antigens [19].

In the literature, the BBK32 protein has also been called P35 [9, 13,20] and 47 kilodalton fibronectin binding (FBP) protein [21].

DETAILED DESCRIPTION OF THE INVENTION

The purpose of the inventors was to test the antigenic potential ofborrelial protein BBK32 and develop a reliable immunoassay method forthe serodiagnosis of early and disseminated LB, especially for theserodiagnosis of erythema migrans. In order to cover all pathogenicborrelial species causing human LB, the inventors sequenced and clonedthe bbk32 from eight isolates of the three pathogenic borrelial species,B. burgdorferi sensu stricto, B. afzelii, and B. garinii. The identitybetween the amino acid sequences of BBK32s in B. burgdorferi sensustricto, B. garinii, and B. afzelii isolates was 71-100%. The respectivevariant BBK32 recombinant proteins were tested in LB serology usingserum samples from patients with early- and late-stage LB. In IgGWestern blotting (WB) or enzyme-linked immunosorbent assay (ELISA), upto 74% and 100% of acute and convalescent samples from 23 patients witherythema migrans (EM) were positive for recombinant BBK32 protein fromB. afzelii. In the serology of disseminated LB, the three variant BBK32antigens cross-reacted. In total, the sensitivity of IgG ELISA reached100%. The results show that the BBK32 proteins are useful serodiagnosticantigens for early and disseminated LB, but in order to cover all therelevant borrelial species variant BBK32 proteins or fragments thereofshould be used in parallel or combined in an immunoassay for LB.

It is therefore an object of the present invention to provide a novelmethod for detecting Borrelia burgdorferi sensu lato infection or thepresence of antibodies against Borrelia species in a body fluid from asuspected infected or vaccinated human or other mammal, in which methodrecombinant BBK32 proteins or their fragments containing antigenicepitopes from one or more Borrelia species are used as antigens in animmunoassay. The method according to the invention can be used for theserodiagnosis of early and late Lyme borreliosis, especially for theserodiagnosis of erythema migrans.

Within the scope of this invention and this specification, a ‘fragment’is intended to mean a recombinantly produced fragment containingantigenic epitopes of the immunogenic protein(s) in question.

In a preferred embodiment according to the invention, recombinant BBK32proteins or their fragments together with any other immunogenic proteinsor their fragments derived from one or more Borrelia species are used asantigens in an immunoassay. Examples of the other immunogenic borrelialproteins which can be used in the method according to the inventioninclude, but are not limited to, outer surface protein A (OspA), OspB,OspC, OspE, OspF, P22, P39, P100, VlsE, DbpA, and/or flagellin,preferably DbpA.

Preferably the recombinant BBK32 proteins, optional other immunogenicproteins, or their fragments are derived from at least two Borreliaspecies selected from the group consisting of B. burgdorferi sensustricto, B. afzelii, and B. garninii, more preferably from all the threeBorrelia species mentioned above.

It is apparent to those skilled in the art that instead of borrelialproteins or their fragments, or together with them, also peptides orpolypeptides (referred here as peptides), i.e. shorter amino acidstretches comprising at least two, usually several amino acids, can beused as antigens in the method according to the invention. It is alsopossible to take a combination of several fragments or peptides fromvarious immunogenic borrelial proteins and to use them as antigens.

The recombinant BBK32 proteins, optional other immunogenic proteins, ortheir fragments are used as antigens either in parallel or combined inan immunoassay. When recombinant BBK32 proteins are used as ‘parallel’antigens, antibodies are measured separately against the recombinantBBK32 antigens from different Borrelia species in the same assay. It isalso possible to combine the recombinant BBK32, other immunogenicborrelial antigens, or their fragments from different Borrelia speciesand measure antibodies against the ‘combined’ BBK32 antigen. For examplein a parallel assay, three recombinant BBK32 protein antigens from threeBorrelia species are preferably used in the same assay. In a combinedassay, preferably three recombinant BBK32 proteins from three Borreliaspecies are combined to form the combined BBK32 protein antigen.

Within the scope of this invention, an ‘immunoassay’ is intended tocover all immunoassay methods known to persons skilled in the art.

The method according to the invention for detecting Borrelia burgdorferisensu lato infection or the presence of antibodies against Borreliaspecies in a body fluid from a suspected infected or vaccinated human orother mammal comprises preferably the steps of

-   -   a) contacting the body fluid with recombinant BBK32 proteins,        optional other immunogenic proteins, or their fragments derived        from one or more Borrelia species under conditions effective to        allow the formation of antigen-antibody complexes; and    -   b) detecting the complexes formed.

The body fluid is preferably a serum, plasma, whole blood, cerebrospinalfluid, or synovial fluid sample.

The conditions effective to allow the formation of antigen-antibodycomplexes as well as the means for detecting the complexes formed arechosen according to the antibodies and other reagents used in the assayand are known to a person skilled in the art.

It is a further object of the invention to provide a diagnostic kituseful for detecting Borrelia burgdorferi sensu lato infection or thepresence of antibodies against Borrelia species in a body fluid from asuspected infected or vaccinated human or other mammal, said kitcomprising in a suitable container

-   -   a) recombinant BBK32 proteins, optional other immunogenic        proteins, or their fragments from one or more Borrelia species        and a detectable label or marker linked to said proteins and/or        their fragments, or    -   b) recombinant BBK32 proteins, optional other immunogenic        proteins, or their fragments from one or more Borrelia species        and a second antibody linked to any detectable label or marker.

Detectable labels or markers and methods to link them to antigens orsecond antibodies are well disclosed in the literature and are alsoknown to persons skilled in the art of immunoassays.

A further object of the invention is an immunoassay method fordiagnosing early and late Lyme borreliosis, especially for theserodiagnosis of erythema migrans, comprising the steps of

-   -   a) contacting a body fluid from a human or other mammal with        recombinant BBK32 proteins, optional other immunogenic proteins,        or their fragments derived from one or more Borrelia species        under conditions effective to allow the formation of        antigen-antibody complexes; and    -   b) detecting the complexes formed.

A still further object of the invention is to provide a novel method fordetecting Borrelia burgdorferi sensu lato infection or the presence ofantibodies against Borrelia species in a body fluid from a suspectedinfected or vaccinated human or other mammal, in which methodrecombinant BBK32 proteins and recombinant decorin binding protein As(DbpAs) from at least two, preferably three, Borrelia species are usedtogether as antigens in an immunoassay. Decorin binding protein A is aborrelial outer surface protein, which has been suggested to act as aspecies-specific serodiagnostic antigen for LB. In a preferredembodiment according to the invention, the recombinant BBK32 proteinsand the recombinant DbpAs are derived from B. burgdorferi sensu stricto,B. afzelii, and B. garinii.

Antibodies to the BBK32 protein seem to appear very early during humanLB. In up to 74% of the patients at acute-phase EM, IgG antibodies toBBK32 were detectable by ELISA and/or WB. At follow-up, after successfulantibiotic treatment, all the patients were anti-BBK32antibody-positive. A recent study reported cloning of the bbk32 from aB. burgdorferi sensu stricto isolate and showed early antibody responsesto the recombinant BBK32 protein during experimental murine borreliosis[9]. Reverse transcriptase-PCR studies have also demonstrated bbk32expression in EM lesions of three patients, indicating that, duringhuman LB, bbk32 is expressed early [20]. However, antibody responses toBBK32 in patients with early local EM have not previously been studied.

With the current LB serology based mainly on flagellin as an antigen,IgM and IgG antibodies are not detectable by ELISA or immunoblot assaysuntil 2-4 or 6-8 weeks after the onset of the disease [6]. During earlylocal LB, the sensitivity of IgM ELISA seldom exceeds 50% [6, 22-24]. Astudy on patients with culture-confirmed EM showed that positiveserology at presentation and the rate of seroconversion correlateddirectly with disease duration [25]. If the EM lesion had emerged lessthan 7 days prior to sampling, only 10% of the patients showedantibodies in ELISA, whereas, of the patients whose EM had occurred 7 to14 days earlier, 58% had detectable antibodies. A recent study reportingon anti-BBK32 antibodies in disseminated LB also showed IgG BBK32antibodies in 84% of patients with EM [13]. However, in these patients,the EM lesion had been present for 2 weeks to 3 months after diseaseonset, suggesting dissemination of LB. In the present series, the timeof occurrence of the EM lesion could not be accurately assessed.However, given the low proportion of seropositivity to flagellin atpresentation of EM (17-26%) and the broad awareness of LB among thegeneral population in regions where LB is endemic in Finland, it can bepresumed that, in most cases, the EM lesions represented early disease.Hence, the present results imply that assessment of IgG, although not ofIgM antibodies, to BBK32 proteins affords a major improvement in theserodiagnosis of early LB. The antibody response to the BBK32 proteinalso seems to precede the humoral response to in vitro-grown microbes(WCL), which do not necessarily synthesize this in vivo-expressedprotein [9, 21].

Only a few studies have evaluated the antigenic properties of the BBK32protein. In these studies, the BBK32 proteins originated from AmericanB. burgdorferi sensu stricto strains [13, 20]. In a preferred embodimentof the present invention variant recombinant proteins from the threepathogenic borrelial species, B. burgdorferi sensu stricto, B. afzelii,and B. garinii, are used in the serodiagnosis of LB, especially in theserodiagnosis of erythema migrans. Recombinant BBK32 originating from alocal B. afzelii isolate appeared to be superior to the other rBBK32proteins for diagnosing EM. This finding agrees with the PCR results ofthe EM skin biopsies, where the majority of the infecting species provedto be B. afzelii. These observations are also in accord with two recentEuropean studies, where over 90% of Borrelia isolates from EM lesionswere B. afzelii, less than 10% B. garinii, and none were B. burgdorferisensu stricto [26, 27]. The present results suggest that in diverseepidemiological situations, for the serodiagnosis of early local EM,variant BBK32 proteins are preferred.

Although the immunoreactivity of EM patient sera to variant BBK32proteins diverged, the three recombinant BBK32 antigens “cross-reacted”in the serology of disseminated LB. Furthermore, the intensities of theserologic responses against variant BBK32 proteins, as measured by ODvalues in ELISA, correlated well. Therefore, these results indicate thatvariant BBK32 proteins may have both specific and common antigenicepitopes. In European epidemiological studies, the most prevalentBorrelia species have been B. afzelii and B. garinii [28], B.burgdorferi sensu stricto occurring infrequently, especially inScandinavia [27, 29]. In cerebrospinal fluid samples of Europeanneuroborreliosis cases, the species predominantly isolated has been B.garinii [30]. The hypothesis that epitope specificity varies in earlyand late LB is in line with the analysis of BBK32 sequences of eightFinnish isolates of B. burgdorferi sensu lato, showing over 90% identitybetween the sequences of B. burgdorferi sensu stricto and B. garinii. Incontrast, the identity between the BBK32 sequences of B. afzelii strainsand of other species was approximately 70%.

In the serodiagnosis of disseminated LB, the BBK32 antigen has beenevaluated in a limited number of patients only, so far [9, 13]. Fikriget al. [9] reported high IgG antibodies to BBK32 in 3 of 3 patients withneuroborreliosis and in 3 of 7 patients with Lyme arthritis. Akin et al.[13] showed IgG responses to BBK32 in 83-92% of 25 Lyme arthritispatients. The preferred embodiment of the present invention with thethree variant BBK32 proteins as antigens improved the sensitivity ofELISA up to 100%. In the serodiagnosis of neuroborreliosis, all but onecase would have been detected irrespective of the origin of the BBK32antigen. Instead, especially in ELISA for Lyme arthritis, use of asingle BBK32 antigen would have left the sensitivity at 80-90%. Theoccasional discrepancies between the results of WB and ELISA may be dueto differences in the orientation of the antigen and/or antigen-antibodycomplex formation.

In summary, the inventors have shown that the BBK32 proteins are usefulantigens for both early and late LB serology. The BBK32 from B. afzeliiproved to be a sensitive antigen of EM already at presentation. Duringthe course of infection, the sensitivity increased being up to 100% inconvalescence samples for EM patients. However, it is evident thatvariant BBK32 proteins should be used either in parallel or combinedwith an immunoassay for LB to cover all the relevant borrelial species,whose prevalence differs regionally in Europe.

The following examples further illustrate the invention without,however, limiting the same.

EXAMPLES

Bacterial strains. Finnish borrelial strains were obtained from theNational Public Health Institute, Turku, Finland. B. burgdorferi sensustricto strain ia (here referred to as Bbia) was isolated from thecerebrospinal fluid of a Finnish patient with neuroborreliosis. Of theB. afzelii strains, A91 and 1082 (referred to as BaA91 and Ba1082) wereisolated from skin biopsy samples of Finnish patients with erythemamigrans EM), and 570 and 600 (referred to as Ba570 and Ba600) wereisolated from ticks. B. garinii strains 40, 46, and 50 (referred to asBg40, Bg46, and Bg50, respectively) were isolated from skin biopsysamples of Finnish patients with EM. The genotypes of culture-positiveBorreliae were confirmed by sequencing a fragment of the flagellin gene[29]. Borreliae were cultivated in BSK-H (Barbour-Stoenner-Kelly) medium(Sigma, USA) at 33° C. in 5% CO₂. The B. afzelii strain SK1 was used inan in-house ELISA for detecting antibodies against borrelial WCL.Escherichia coli host cells for cloning and for expression ofrecombinant proteins were INFαF (Invitrogen, Netherlands) and BL21(Amersham Pharmacia Biotech, Sweden), respectively.

DNA purification. Borrelial genomic DNA was purified with a DneasyTissue Kit (Qiagen, Germany). Purified DNA was used in PCR and incloning experiments. Plasmid DNA was purified with a QIAprep-spinplasmid kit (Qiagen, USA).

PCR and DNA sequencing. A PCR-based approach was employed to amplify andsequence the bbk32 alleles from eight different isolates of B.burgdorferi sensu lato. Primers for bbk32 sequencing were designed onthe basis of published bbk32 sequences (Table 1). Several primer pairswere designed and tested to ensure that the entire coding sequence ofthe bbk32 was obtained. To eliminate possible errors caused byTaq-polymerase, the two strands for each bbk32 were sequencedindependently at least twice. Expression primers for each strainencoding the mature portion of the BBK32 protein after cysteine at thesite of posttranslational acylation were chosen from the sequencesanalyzed. For each borrelial strain, the bbk32 sequences were generatedby PCR amplification of B. burgdorferi genomic DNA. Approximately 1 ngof template DNA was used in standard PCR conditions: 30 cycles of 94° C.denaturing for 1 min, 50° C. annealing for 1 min, and 72° C. extensionfor 1 min 30 s with AmpliTaqGold DNA polymerase (Perkin Elmer, USA). ThePCR-amplified full-length or partial bbk32s were cloned to the pCR2.1-TOPO vector (Invitrogen, Netherlands) for sequencing. DNA sequencingwas performed at the Core Facility of the Haartman Institute, Universityof Helsinki, with DyePrimer (T7, M13Rev) cycle sequencing kit (AppliedBiosystems Inc., USA). Sequencing reactions were run and analyzed by theautomated sequencing apparatus model 373A (Applied Biosystems Inc.,USA). DNA and protein sequences were analyzed with Lasergene software(DNASTAR, USA). TABLE 1 Primers used for PCR amplification of the bbk32genes No. Species Primer 5′-3′ Location Source 1 B. buredorferi CAC CCTCTT GAT AGC ACT TA −203--184 B31(AF000788) 2 sensu stricto CTT TAA AGGAGA GAA AGC ATG −18-3  Bbia(AF472532) 3 CCG GAT CCG ATT TAT TCA TAA GATATG AAA T 60-82 Bbia 4 GCA ATC TGA GAC TAG AAA AG 329-348 Bbia 5 TGC AGTCTT TAC ACT TAC TT 879-860 Bbia 6 CCC TCG AGA TTA GTA CCA AAC GCC ATT1084-1065 Bbia 7 ACA TAT TAT GTA GCC TGT TTT A 1122-1101 B31 2 B.garinii CTT TAA AGG AGA GAA AGC ATG −18-3  Bbia 3 CCG GAT CCG ATT TATTCA TAA GAT ATG AAA T 60-82 Bg40(AF472529) 4 GCA ATC TGA GAC TAG AAA AG329-348 Bg40 5 TGC AGT CTT TAC ACT TAC TT 879-860 Bg40 8 CCC TCG AGA GTACCA AAT GCC ATT CT 1084-1064 Bg40 7 ACA TAT TAT GTA GCC TGT TTT A1122-1101 B31 2 B. afzelii CTT TAA AGG AGA GAA AGC ATG −18-3  Bbia 9CCG GAT CCG ATT TAT TCA TAA GAG ATG AAA T 57-79 BaA91(AF472525) 10 TGAGCA TAA AAG GAT GCT TC 369-387 BaA91 11 GCA GTC CTT GCA CTC ACT 855-838BaA91 12 CCC TCG AGC AAA GAT TAG TAC CAA ACA C 1065-1046 BaA91 7 ACA TATTAT GTA GCC TGT TTT A 1122-1101 B31Restriction enzyme sites for BamHI and XhoI in expression primers areunderlinedPrimers 2 and 7 were used in all strainsPrimers 3, 4, and 5 were used both in B. burgdorferi sensu stricto andB. garinii PCR amplifications

Cloning and expression of recombinant BBK32. For expression of therecombinant BBK32 (rBBK32), glutathione S-transferase (GST) fusionprotein constructs were generated. The PCR-amplified DNA encoding themature portion of BBK32 was cloned into the pCR 2.1-TOPO plasmid(Invitrogen, Netherlands). The recombinant plasmid was purified anddigested with BamHI and XhoI restriction enzymes. The cleaved bbk32 wasthen ligated to a similarly digested pGEX4T-1 expression plasmid(Amersham Pharmacia Biotech, Sweden) and transformed into E. coli BL21host cells. The expression of recombinant GST-BBK32 protein wasgenerated according to the manufacturer's instructions (AmershamPharmacia Biotech, Sweden). The expression and purity of the GST-rBBK32fusion protein was confirmed by sodium dodecyl sulfate-polyacrylamidegel electrophoresis (SDS-PAGE).

Western blotting. GST-rBBK32s originating from Bbia, BaA91, and Bg40(referred to here as rBBK32_(Bbia), rBBK32_(BaA91), or rBBK32_(Bg40),respectively) were fractionated in 10% SDS-PAGE and transferred to anitrocellulose membrane (BioRad, 0.2 μm pore size, USA) by semi-drytransfer with 40 mM glycine-50 mM Tris (pH 9.0)-0.375% (w/v) SDS-20%(v/v) methanol buffer. Equal amounts of each GST-rBBK32 were used forone 7-cm-wide nitrocellulose membrane. Two-mm strips of thenitrocellulose membranes were soaked in 0.1% Tween 20, 0.9% NaCl. Serumsamples were diluted in 0.1% Tween 20, 0.9% NaCl, 0.1 g/l fat-freebovine milk powder (Valio, Finland). Samples were incubated at 1:100dilution for 2 h. After four buffer rinses, the Western blots wereincubated with alkaline phosphatase-conjugated rabbit anti-human IgG(Jackson Immuno Research Laboratories Inc., USA) at 1:5000 for 2 h.After washing, the bands were visualized with5-bromo-4-chloro-3-indolylphosphate nitro blue tetrazolium (SigmaChemical Co., USA). The reaction was terminated 10-15 min later bywashing with distilled water. The WB results were analyzed with MacBAS2.5 (Fuji, Japan) software, and the cut-off for a positive IgG WB resultwas defined as the mean+3 standard deviations (SD) of the values ofhealthy blood donors. For detection of GST, monoclonal anti-GSTantibodies (Sigma, USA) were used.

ELISA. ELISA analyses for anti-flagellin antibodies were done asdescribed earlier [31]. Briefly, IgG antibodies against B. burgdorferiwere measured with a commercial flagellin-based ELISA kit (Dako,Denmark) modified by titrating the antibodies. Sera were dilutedserially in three-fold steps for the test and applied to the plates forovernight incubation. The bound antibodies were detected withbiotin-labeled goat anti-human IgG (Zymed, USA). An end-point titer wasobtained at an optical density level determined by a cut-off controlprovided by the kit. The titer limit for a positive IgG antibody levelwas 500. The cut-off control material conformed with the level of themean+3 SD of the reference population living in central Finland, an areawith low prevalence of LB [31].

For ELISA assays measuring anti-BBK32 antibodies, the wells in amicrotiter plate were coated with 100 μl (2 μg/ml) of variant BBK32recombinant proteins overnight. After washing, 100 μl of diluted serumsamples were added to the wells and incubated overnight. Serum sampleswere diluted 1:10 (EM) or 1:100 (neuroborreliosis and Lyme arthritis) in5 mg/ml bovine serum albumin (BSA) in 0.155 M NaCl-0.04% Tween 20 buffer(BSA-NaCl-Tween). After washing, the wells were incubated with alkalinephosphatase-conjugated rabbit anti-human IgG or IgM (Jackson ImmunoResearch Laboratories Inc., USA) 1:5000 in BSA-NaCl-Tween for 2 h. Thereactions were visualized with 4-nitrophenylphosphate (BoehringenMannheim GmbH, Germany) 1 mg/ml in diethanolamine buffer pH 10.0. Theoptical density (OD) measurements were made after 10 to 20 minutes atwavelength 405 nm using a Multiscan photometer (Thermo Labsystems,Finland).

Samples. 1. For serological analyses, human serum samples were collectedfrom Finnish patients with culture- or PCR-positive EM, withneuroborreliosis (NB), and with Lyme arthritis (LA). Samples werecollected from EM patients at diagnosis (acute) and 1 to 3 months aftertreatment (convalescent). Of the 23 patients with EM, genotyping by PCRanalysis [29] showed B. afzelii in 17, and B. garinii in 4 of the skinbiopsies. In two biopsies, genotyping was not feasible. In the patientswith disseminated LB, the clinical manifestations agreed with the CDCcriteria for LB [32]. The clinical diagnosis was confirmed in ELISA bydemonstrating serum antibodies (and CSF anti-flagellin antibodies in NBpatients) against flagellin and B. burgdorferi WCL. Serum samples frompatients with syphilis, Epstein-Barr virus (EBV) infection, systemiclupus erythematosus (SLE), rheumatoid factor (RF) positivity,anti-streptolysin (ASO) positivity, and sera from healthy blood donorswere used as controls.

2. Serum samples were also collected from patients with clinicallydocumented or culture- or PCR-confirmed EM from Germany, Slovenia, andUSA. German samples were collected in Northern Bavaria as part of aregional study on LB [35]. Sera were collected from 22 patients withphysician-diagnosed EM at the time of diagnosis and during theconvalescence phase. The clinical diagnosis of EM was confirmed byespecially trained physicians. In addition, from 10 US patients withculture-positive EM sera were collected at the time of diagnosis andduring convalescence 3-4 weeks after treatment [36]. Serum samples wereavailable from 20 Slovenian patients in the acute stage of EM. Two outof 9 Slovenian patients from whom skin biopsies were taken wereculture-positive (B. burgdorferi sensu lato) [37]. All patients with EMwere treated with oral antimicrobials.

Serum samples from 40 Finnish healthy blood donors were used as negativecontrols and to define the cut-off value for all ELISAs (cut-off=meanplus 3 SD).

Nucleotide sequence accession numbers. The nucleotide sequences of thebbk32 were submitted to GenBank under accession numbers AF472525 for B.afzelii A91 (SEQ ID NO. 1), AF472527 for B. afzelii 1082 (SEQ ID NO. 5),AF472526 for B. afzelii 570 (SEQ ID NO. 3), AF472528 for B. afzelii 600(SEQ ID NO. 7), AF472529 for B. garinii 40 (SEQ ID NO. 9), AF472530 forB. garinii 46 (SEQ ID NO. 11), AF472531 for B. garinii 50 (SEQ ID NO.13), and AF472532 for B. burgdorferi sensu stricto ia (SEQ ID NO. 15).

Statistical analyses. The Microsoft Excel 2000 program (Microsoft, USA)was used for calculations of standard statistics.

Results

Sequence analysis of BBK32 in the Finnish borrelial isolates. Thededuced amino acid sequences of BBK32_(BaA91), BBK32_(Ba1082),BBK32_(Ba570), BBK32_(Ba600), BBK32_(Bg40), BBK32_(Bg46), BBK32_(Bg50),and BBK32_(Bbia) contained 352 to 360 residues. The sequences of all theBBK32 proteins revealed a hydrophobic leader sequence of 18 to 19residues and a phenylalanine-X-Y-cysteine motif, consistent with alipoprotein. Characteristically for borrelial lipoproteins, the greaterpart of the mature portion of the BBK32 protein was hydrophilic (datanot shown). The BBK32 leader sequences in the B. garinii strains and theB. burgdorferi sensu stricto strain were identical, but differed bythree amino acids from the leader sequences in the identical B. afzeliistrains. The inter-species identity of the deduced amino acid sequencesof the BBK32 proteins ranged from 71 to 95% (FIG. 1). The differences inthe amino acid sequences were distributed evenly along the sequence. Theidentity of the BBK32 amino acid sequences within the borrelialsubspecies ranged from 94 to 100%. The calculated molecular mass of themature BBK32 proteins (without putative lipid acylation) ranged from38.7 to 39.5 kDa.

Sequence analysis of BBK32 in B. afzelii strains. The inventorssequenced the bbk32 from two human (BaA91 and Ba1082) and two tickisolates (Ba570 and Ba600). The identity of the deduced amino acidsequences was from 99 to 100%. The BBK32 sequences of the two human B.afzelii isolates were identical. In the GenBank search, one bbk32sequence from B. afzelii was found. The BBK32 sequence of the ACA1strain (AF213179) is only partial, corresponding to the sequence betweenamino acid positions 34 and 336 of the deduced sequence ofBBK32_(BaA91). In the matching regions of the four studied BBK32sequences from B. afzelii and the partial BBK32_(ACA1), sequence,identity was from 99 to 100%.

Sequence analysis of BBK32 in B. garinii strains. The deduced amino acidsequences of BBK32_(Bg40) and BBK32_(Bg50) were identical, andBBK32_(Bg46) was 94% identical with them. In the GenBank search, onepartial bbk32 sequence from B. garinii strain Ip90 (AF213178) was found.This sequence matched the BBK32_(Bg40) sequence between amino acidresidues 35 and 343, but in the BBK32_(Ip90) sequence a six amino aciddeletion was observed from positions 201 to 206. Sequence identity inthe corresponding regions of the BBK32 sequences from B. garinii strainsin this study and the partial BBK32_(Ip90) sequence was from 92 to 93%.

Sequence analysis of BBK32 in B. burgdorferi sensu stricto strains. Thebbk32 sequence from the local strain Bbia was compared with twosequences published in the GenBank. The bbk32 sequence of the B31 strain(AE000788) was complete and that of strain N40 (U82107) was a partialsequence, lacking the first 86 amino acids. The BBK32 amino acidsequences from Bbia and B31 were 96% identical. Identity in thecorresponding regions of BBK32 sequences of Bbia, B31, and N40 was from91 to 94%. In the BBK32_(B31) sequence there was a six amino aciddeletion at positions 201 to 206 of BBK32_(Bbia). In the same region,three tyrosine residues were deleted from the BBK32_(N40) sequence.

Western blotting. In IgG Western blots using rBBK32 proteins from B.afzelii (BaA91) as antigen with serum samples from Finnish patients withculture- or PCR-positive EM, 10/15 (67%) at the acute and 15/15 (100%)at the convalescent phase were positive FIG. 2). With rBBK32_(Bg40) asan antigen, 4/15 and 8/15, and with rBBK32_(Bbia), 7/15 and 3/15 werepositive at the acute and convalescent phases, respectively. Of the 10control patients (five with syphilis and five healthy blood donors), onewith syphilis had a weak antibody response to rBBK32_(BaA91) (data notshown). In ELISA for anti-fiagellin antibodies (Dako, Denmark), 6/15patients had IgG or IgM antibodies at the acute or convalescent phase(FIG. 2). One patient had IgM antibodies in the in-house ELISA (WCL asantigen).

In another WB experiment, serum samples from 10 patients with NB and 10with LA were analyzed for the presence of anti-BBK32 IgG antibodies. All20 patients with disseminated LB reacted positively with the threerBBK32 proteins as determined with the MacBAS program, using the blooddonors' mean+3 SD as a cut-off value (Table 2). Minor differences wereobserved in the immunoreactivity of serum samples against differentrBBK32 proteins. Specifically, in four serum samples, weakerreactivities were observed against rBBK32_(BaA91) than against the otherrBBK32s (data not shown). A few of the control samples were lowpositives (Table 2). None of the patient or control sera recognized pureGST (data not shown). TABLE 2 IgG Western blotting reactivity againstrecombinant BBK32 proteins from B. afzelii (BaA91), B. garinii (Bg40),and B. burgdorferi sensu stricto (Bbia) of LB patients and controls. Theintensities of WB bands were analyzed with MacBas 2.5 software. No. ofpositive samples/ Patient/ no. of samples recombinant BBK32 controlgroup BaA91 Bg40 Bbia Neuroborreliosis 10/10 10/10 10/10 Lyme arthritis10/10 10/10 10/10 Syphilis 1/5 0/5 2/5 Positive for RF* 0/5 0/5 1/5Blood donors 0/5 0/5 0/5*RF, rheumatoid factor

ELISA. 1. Serum samples from Finnish LB patients and controls weretested in IgG ELISA, using all three rBBK32 proteins individually asantigens. In patients with NB, 14/14, 13/14, and 14/14 samples werepositive when rBBK32_(BaA91), rBBK32Bg₄₀, and rBBK32_(Bbia),respectively, were used as antigens. In serum samples from patients withLA, these figures were 12/15, 11/15, and 14/15, respectively. In total,14 of 14 (100%) samples from patients with NB, and 15 of 15 (100%)samples from patients with LA were positive for one or more rBBK32proteins (FIG. 3). The ELISA OD values of NB patients correlated wellbetween variant BBK32 proteins as antigens, the correlation coefficientsbeing 0.91, 0.78, and 0.83, between BBK32 from BaA91 and Bg40, BaA91 andBbia, and Bg40 and Bbia, respectively. In LA patients, the respectivecorrelation coefficients were 0.89, 0.90, and 0.93.

Serum samples from patients with EM were analyzed in both IgG and IgMELISA. In IgG ELISA with rBBK32_(BaA91) as an antigen, 17/23 (74%)samples taken at the acute and 15/23 (65%) at the convalescent phasewere positive (FIG. 4). On the other hand, when rBBK32Bg40 andrBBK32_(Bbia) were used as antigens, 6/23 (26%) and 5/23 (22%) of acutesamples, and 7/23 (30%), and 4/23 (17%) of convalescent samples,respectively, were positive. Of the control samples, 2/10 with syphilis,2/8 RF-positive, 4/10 EBV-positive, and 2/20 healthy blood donor samplesshowed low positive OD values. In IgM ELISA with samples from EMpatients, 4 to 13% of the acute or convalescent samples were positive,depending on the rBBK32 antigen used.

In the ELISA tests for anti-flagellin antibodies, 6/23 (26%) patientshad IgG antibodies in either acute or convalescent samples. In IgM ELISAassays for anti-flagellin antibodies, 4/23 (17%) were positive.

2. Antibodies in EM patients from different countries. At presentationof EM, 65 of the 75 (87%) patients Finnish, German, Slovenian, andAmerican patients included) had IgG antibodies to one or more variantsof rBBK32, 29/75 (39%) to flagella, and 29/75 (39%) to the IR₆ peptideantigen (Table 3, FIG. 5). In samples from different regions, reactivityto the rBBK32 variants diverged. The majority of patients from Finlandhad antibodies to rBBK32 from B. afzelii, whereas, in the German andSlovenian sera, the most sensitive antigen was rBBK32 from B. garinii.All 10 samples from the USA reacted positively with BBK32 from B.burgdorferi sensu stricto and from B. garinii.

In the different regions, the proportion of patients with IgManti-flagella antibodies at the time of diagnosis varied from 13% to45%. Of the total of 75 patients, 29% had IgM anti-flagella antibodies.IgM and/or IgG anti-flagella antibodies were detected in 46% of thepatients.

Antibodies in the convalescent phase of EM patients. Forty of the 55patients (73%) in the convalescent phase had IgG antibodies to one ormore rBBK32s, 25/55 (45%) to flagella and 19/55 (35%) to the IR₆peptide. The pattern of seropositivity to variant rBBK32s in theconvalescent and the acute sera was similar. In the convalescencesamples from Finland and Germany, the overall rate of IgG antibodypositivity

BBK32 and from that at diagnosis (Table 3, FIG. 5).

The study with sera from epidemiologically diverse regions providessupporting evidence that the BBK32 proteins may be useful antigens in EMserology. At presentation of EM, the sensitivity of the BBK32 ELISAsappeared better than the anti-flagella or the new anti-IR₆ tests. TABLE3 Number of positive sera (%) for any of the three BBK32 variantantigens (BBK32 - all), IR₆ peptide antigen, and for flagella antigen(Dako) in patients with EM from Finland (FIN), Germany (GER), USA, andSlovenia (SLO). AT DIAGNOSIS AT CONVALESCENCE FIN GER USA SLO TOTAL FINGER USA TOTAL Antigen n = 23 n = 22 n = 10 n = 20 n = 75 n = 23 n = 22 n= 10 n = 55 BBK32 - all* 17 (74%) 21 (95%) 10 (100%) 17 (82%) 65 (87%)15 (65%) 15 (68%) 10 (100%) 40 (73%) IR6* 7 (30%) 7 (32%) 4 (40%) 11(55%) 29 (39%) 6 (26%) 6 (27%) 7 (70%) 19 (35%) Flagella* 6 (26%) 9(41%) 4 (40%) 10 (50%) 29 (39%) 6 (26%) 11 (50%) 8 (80%) 25 (45%)Flagella** 3 (13%) 8 (36%) 2 (20%) 9 (45%) 22 (29%) 4 (17%) 7 (32%) 8(80%) 19 (34%) Flagella*** 6 (26%) 13 (59%) 4 (40%) 12 (60%) 35 (46%) 8(35%) 15 (68%) 10 (80%) 33 (60%)*IgG class antibodies;**IgM class antibodies;***IgG and/or IgM antibodies

Antibodies to BBK32 in cerebrospinal fluid (CSF) samples. Antibodies toBBK32 have also been measured in the cerebrospinal fluid (CSF). CSFsamples were obtained from 85 patients who had been treated forneuroborreliosis. The clinical diagnosis of neuroborreliosis was basedon the clinical guidelines for diagnosis presented by the Centers forDisease Control and Prevention, USA. As a control assay, CSF antibodiesto purified intact flagella of B. afzelii (Dako, Denmark) weredetermined. The CSF samples had also been studied for pleocytosis. Basedon the anti-flagella antibodies and CSF findings, the patients weredivided into three groups: confirmed (elevated anti-flagella IgGantibodies and pleocytosis in CSF), probable (elevated anti-flagella IgGantibodies but no pleocytosis in CSF), and possible (no anti-flagellaIgG antibodies in CSF but either serum anti-flagella antibodies orpleocytosis in CSF) neuroborreliosis. In the classification, CSFanti-flagella antibodies were defined as significant if the IgGanti-flagella antibody titer in the CSF was higher than that of theserum divided by 400 (the approximate serum IgG/CSF IgG ratio in healthypersons). According to this definition, CSF anti-flagella antibodytiters >20 were regarded as significant. Furthermore, patients weredefined to have neuroborreliosis of short duration if the symptoms hadlasted less than 3 months and of long duration if the symptoms hadlasted longer than 3 months. As controls, we used CSF samples from 14patients with syphilis, from 32 patients with other neurologicaldiseases such as confirmed viral meningitis or convulsions/epilepsy, andfrom 20 patients without any proven infection. All the CSF samples fromthe-controls were negative for anti-flagella antibodies.

IgG ELISAs were performed with rBBK32 antigens, including three variantsoriginating from B. burgdorferi sensu stricto, B. afzelii, and B.garinii, as described above. For rBBK32 ELISA, each well was coatedovernight at +4° C. with 200 ng of protein, rBBK32 diluted in 3 M urea.The CSF samples were used at 1:10 dilution in NaCl-0.04% Tween 20 buffer(BNT). Samples of 20 CSF controls without proven infection were used todefine the cutoff value (mean plus 3 SD).

Of all the 85 patients, 70% had CSF antibodies to rBBK32, whereasanti-flagella antibodies were observed in 53% of the patients. Of the 40patients with negative or borderline anti-flagella antibodies (possibleneuroborreliosis), 43% had anti-BBK32 antibodies (FIG. 6).

Of the 60 patients with duration of the disease <3 months, 73% had CSFantibodies to rBBK32. Of the 25 patients with disease of longer duration(>3 months), 58% had antibodies to rBBK32, mostly at low level (FIG. 7).In the patients with a longer duration of the disease, the antibodylevels to rBBK32 in the CSF were lower than in the patients with a shortduration (p<0.012) (FIG. 7).

Recombinant BBK32 fragment as an antigen. In another experiment, a91-amino acid hydrophilic fragment of the BBK32 protein (from B.afzelii) (fragment) was initially cloned and expressed as a recombinantfusion protein (GST-fusion protein), purified and subsequently tested asan antigen in ELISA. As a comparison, antibodies to the BBK32 wholeprotein (GST-fusion protein) and to a commercial whole cell lysateantigen (Institut Virion/Serion GmbH, Germany) were assessed. The serumsamples were from culture- or PCR-positive EM patients (n=23) or frompatients with Lyme arthritis (n=7), neuroborreliosis (n=7) oracrodermatitis chronica atrophicans (n=3) and from control patients(syphilis, rheumatoid factor positivepatients, salmonella-,yersinia-infection, anti-streptolysin positive patients). In theserology of disseminated borreliosis, the performance of the BBK32fragment in ELISA was as good as the BBK32 whole protein and whole celllysate antigen but in the serology of erythema migrans, ELISA with theBBK32 fragment performed better than the control assays (Table 4). TABLE4 IgG IgG Virion, Virion, BBK32 fragment IgG IgM EM I (n = 23) Positive4 10 5 7 Borderline 3 5 Negative 19 13 15 11 EM II (n = 23) Positive 612 5 6 Borderline 3 6 Negative 17 11 15 11 Syfilis (n = 5) Positive 1 13 1 Borderline 1 2 Negative 4 3 2 2 RF+ (n = 5) Positive 1 2 2 1Borderline 1 Negative 4 3 3 3 Salmonella Positive (n = 5) Borderline 1Negative 5 5 5 4 Yersinia Positive 1 1 (n = 5) Borderline 1 Negative 5 44 4 AST+ (n = 5) Positive 1 Borderline 1 1 Negative 4 5 4 4 ACA (n = 3)Positive 3 3 3 2 Borderline 1 Negative NB (n = 7) Positive 6 5 6 1Borderline 4 Negative 1 2 1 2 LA (n = 7) Positive 7 6 7 4 Borderline 1Negative 1 2

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Identities of deduced amino acid sequences of BBK32 among theisolates of Finnish B. burgdorferi sensu stricto (Bbia), B. garanii(Bg40, Bg46, and Bg50), and B. afzelii (BaA91, Ba1082, Ba570, andBa600). The identities (%) were calculated from the sequences of theentire proteins including the leader peptides with Multiple sequencealignment, Jotun Hein method, Lasergene software.

FIG. 2. Evaluation of sensitivity of BBK32 in IgG Western blotting (WB)for serodiagnosis of early Lyme borreliosis. Serum samples werecollected from culture or PCR-positive patients with erythema migrans atdiagnosis (acute) and 1-3 months after antibiotic treatment(convalescent). Immunoreactivity was assessed by densitometry withMacBas 2.5 software. The cut-off for positive WB was defined as the meanvalue plus 3 SD of 5 healthy blood donors. In IgM and IgG anti-flagellinELISA (Dako, Denmark), the cut-off value was based on the mean OD plus 3SD of healthy controls. Ba=B. afzelii; Bg=B. garinii; ND=not performed.+,−=positive or negative WB or ELISA, respectively, by the methodindicated.

FIG. 3. IgG ELISA OD values with recombinant BBK32 as an antigen from B.afzelii (panel A), B. garinii (panel B), and B. burgdorferi sensustricto (Bbia, panel C) with serum samples from patients withneuroborreliosis (NB) or Lyme arthritis (LA). Control samples were frompatients with syphilis (SY), systemic lupus erythematosus (SLE),Epstein-Barr virus (EBV) infection, positive rheumatoid factor (RF+),positive for anti-streptolysin (ASO), and samples from healthy blooddonors (BD). The cut-off level (mean+3 SD of BD samples) is indicatedwith a line.

FIG. 4. IgG ELISA OD values with recombinant BBK32 as an antigen from B.afzelii (panel A), B. garinii (panel B), and B. burgdorferi sensustricto (Bbia, panel C) with serum samples from erythema migranspatients at the acute (EM I) and convalescent (EM II) phases. Controlsamples were from patients with syphilis (SY), Epstein Barr virus (EBV)infection, positive rheumatoid factor (RF+), and samples from healthyblood donors (BD). The cut-off level (mean+3 SD of BD samples) isindicated with a line.

FIG. 5. ELISA OD/cut-off values of patients with erythema migrans fromFinland (FIN), Germany (GER), USA, and Slovenia (SLO). Serum sampleswere drawn at diagnosis (a) and after antibiotic treatment inconvalescence (c). IgG antibodies to rBBK32 from B. afzelii (BBK32-afz),B. garinii (BBK32-gar), B. burgdorferi sensu stricto (BBK32-sensustricto), and to IR₆ peptide were assessed. Control samples (CO) werefrom 40 healthy blood donors. The level of positivity for OD/cut-offvalues (>1) is indicated with a horizontal line.

FIG. 6. IgG antibodies LISA) to the recombinant BBK32 in the CSF ofpatients with confirmed, probable or possible neuroborreliosis, and ofcontrols, including patients with syphilis, other neurological diseases,and with no proven infection. The level of positivity for OD/cutoffvalues (>1) is indicated by horizontal lines. The highest OD value ofindividual CSF sample with the BBK32 variants in ELISAs was used in theanalyses.

FIG. 7. IgG antibodies to the recombinant BBK32 in the CSF of patientswith duration of neurologic symptoms <3 months (acute) and >3 months(chronic). The level of positivity for the OD/cutoff values (>1) isindicated by a horizontal line. The highest OD value of individual CSFsample with the BBK32 variants in ELISAs was used in the analyses.

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1. A method for detecting Borrelia burgdorferi sensu lato infection orthe presence of antibodies against Borrelia species in a body fluid froma suspected infected or vaccinated human or other mammal, characterizedin that recombinant BBK32 proteins or fragments thereof derived from atleast two Borrelia species selected from the group consisting of B.burgdorferi sensu stricto, B. afzelii, and B. garinii are used asantigens in an immunoassay.
 2. The method according to claim 1 for theserodiagnosis of early and late Lyme borreliosis.
 3. The methodaccording to claim 2 for the serodiagnosis of erythema migrans.
 4. Themethod according to claim 1, characterized in that recombinant BBK32proteins or their fragments and any other immunogenic protein(s) ortheir fragments are used as antigens in an immunoassay.
 5. The methodaccording to claim 4, characterized in that recombinant BBK32 proteinsor their fragments and recombinant decorin binding protein As (DbpAs) ortheir fragments are used an antigens in an immunoassay.
 6. The methodaccording to claim 1, wherein the recombinant BBK32 proteins, optionalother immunogenic proteins, or their fragments are derived from B.burgdorferi sensu stricto, B. afzelii, and B. garinii.
 7. The methodaccording to claim 6 wherein the recombinant BBK32 proteins, optionalother immunogenic proteins, or their fragments are used as antigenseither in parallel or combined in an immunoassay.
 8. The methodaccording to claim 7 wherein recombinant BBK32 proteins, optional otherimmunogenic proteins, or their fragments are used together withborrelial peptides.
 9. The method according to claim 1, comprising thesteps of a) contacting the body fluid with recombinant BBK32 proteins,optional other immunogenic proteins, or their fragments derived from atleast two Borrelia species selected from the group consisting of B.burgdorferi sensu stricto, B. afzelii, and B. garinii, under conditionseffective to allow the formation of antigen-antibody complexes; and b)detecting the complexes formed.
 10. The method according to claim 9,characterized in that the body fluid is a serum, plasma, whole blood,cerebrospinal fluid, or synovial fluid sample.
 11. A diagnostic kituseful for detecting Borrelia burgdorferi sensu lato infection or thepresence of antibodies against Borrelia species in a body fluid from asuspected infected or vaccinated human or other mammal, said kitcomprising in a suitable container a) recombinant BBK32 proteins ortheir fragments and optionally other immunogenic proteins or theirfragments from at least two Borrelia species selected from the groupconsisting of B. burgdorferi sensu stricto, B. afzelii, and B. garinii,and a detectable label or marker linked to said proteins and/orfragments, or b) recombinant BBK32 proteins or their fragments andoptionally other immunogenic proteins or their fragments from at leasttwo Borrelia species selected from the group consisting of B.burgdorferi sensu stricto, B. afzelii, and B. garinii, and a secondantibody linked to any detectable label or marker.
 12. The diagnostickit according to claim 11 for the serodiagnosis of early and late Lymeborreliosis, especially for the serodiagnosis of erythema migrans. 13.The diagnostic kit according to claim 11, wherein the kit comprisesrecombinant decorin binding protein As (DbpAs).
 14. An immunoassaymethod for diagnosing early and late Lyme borreliosis comprising thesteps of a) contacting a body fluid from a human or other mammal withrecombinant BBK32 proteins or their fragments and optionally with otherimmunogenic proteins or their fragments derived from at least twoBorrelia species selected from the group consisting of B. burgdorferisensu stricto, B. afzelii, and B. garinii, under conditions effective toallow the formation of antigen-antibody complexes; and b) detecting thecomplexes found.
 15. The immunoassay method according to claim 14 forthe serodiagnosis of erythema migrans.
 16. The immunoassay methodaccording to claim 14, wherein recombinant BBK32 proteins andrecombinant decorin binding protein As (DbpAs) are used as antigens.